Heparin enhanced process for separating antihemophilic factor (Factor VIII) and fibronectin from cryoprecipitate

ABSTRACT

A process is provided for preparing fibronectin and antihemophilic factor (Factor VIII) concentrates from a blood plasma cryoprecipitate.

This is a continuation of application Ser. No. 07/779/937, filed Oct.21, 1991 now abandoned, which is a division of application Ser. No.07/363,968 filed Jun. 9, 1989, now abandoned.

FIELD OF THE INVENTION

This invention relates to an improved process for preparing fibronectinand antihemophilic factor (Factor VIII) concentrates from blood plasmacryoprecipitate.

BACKGROUND OF THE INVENTION

Blood is made up of red blood cells (erythrocytes) carried in a solutioncalled "plasma." Blood plasma includes numerous proteins which areuseful for treating humans for various medical indications. Two examplesof such proteins are fibronectin, also known as cold-insoluble globulin(CIg) and anti-hemophilic factor (AHF or Factor VIII).

Fibronectin is a multi-functional glycoprotein present in plasma at aconcentration of approximately 300 micrograms per milliliter. Some ofits biological properties include adhesion of cells to surfaces andbinding to collagen, fibrin, and heparin. Reduced levels of fibronectinhave been found in patients with trauma, burn injury, sepsis and severemalnutrition. Replenishment of fibronectin levels has been correlatedwith clinical improvement in such patients.

Fibronectin has been found to have a molecular weight of 440,000 daltonsand to consist of two nearly identical 220,000-dalton subunitpolypeptides. Fibronectin compositions or concentrates can be made up ofvarying ratios of both the dimer (the 440,000-Dalton unit) and themonomer (the 220,000-dalton unit). It has been suggested that theactivity of a fibronectin composition is relatively higher when theproportion of dimer to monomer is relatively higher.

Factor VIII, which is associated with the coagulation or clotting ofblood, is present at deficient levels in certain individuals, or isabsent. For example, persons who have a deficiency (or absence) ofantihemophilic factor (AHF or Factor VIII), i.e., persons suffering fromhemophilia A, have blood which either fails to clot or clots only afterlonger periods of time than the time required for clotting in a personwho has a normal level of Factor VIII.

In the past, persons suffering from hemophilia A (hemophiliacs) weretreated by transfusing them with whole blood or blood plasma. Morerecently, however, Factor VIII concentrates have become available foradministration to such persons. Such Factor VIII concentrates areproduced by fractionating blood plasma into various components andrecovering the components separately, including the Factor VIIIcomponent. Some processes for producing Factor VIII concentrate havebeen based on a discovery by Poole et al (Nature, Vol. 203, p. 312,1964) that the precipitate remaining after plasma is frozen and thenthawed, i.e., the cryoprecipitate, contains Factor VIII in aconcentrated form and excludes various other protein fractions. It wasdiscovered that, in addition to Factor VIII, the cryoprecipitate alsoincludes the major portion of the fibronectin component of plasma.

Work progressed over the years to perfect the separation of Factor VIIIfrom other proteins in the cryoprecipitate, including the fibronectincomponent, so that the resulting products would incorporate increasedconcentrations of Factor VIII relative to the other proteins present inthe plasma.

One area in which a substantial amount of work has been done inproduction of Factor VIII is based on the use of polyethylene glycol(PEG) to precipitate Factor VIII from other proteins in an aqueouscryoprecipitate solution. For example, U.S. Pat. No. 3,652,530, whichissued on Mar. 28, 1972 to A. J. Johnson, discloses a process forpreparing a Factor VIII concentrate by fractionating cryoprecipitatewith PEG at a relatively lower concentration to precipitate fibrinogenand other proteins, and then increasing the PEG concentration toprecipitate Factor VIII. There is no disclosure of the fate of thefibronectin.

U.S. Pat. No. Re. 29,698 to Fekete et al discloses a process forproduction of Factor VIII (AHF) by which heparin is added to acryoprecipitate solution, along with PEG, to provide increased yields ofprecipitated Factor VIII. It is disclosed that the amount of heparinemployed during the fractionation step can vary, with the optimumconcentration being 1 unit of heparin per ml of the plasma solution,whereas concentrations of heparin greater than about 10 units per ml areto be avoided as dangerous.

It is desired to provide a process for producing fibronectin and FactorVIII which results in increased purity, higher concentration, andenhanced yields of both proteins. Further, it is desired that theprocess provide a fibronectin concentrate comprising a maximized ratioof fibronectin dimer to monomer.

SUMMARY OF THE INVENTION

A process is provided in accordance with this invention for producingfibronectin and Factor VIII concentrates of high purity, yield andconcentration from blood plasma. The process includes the steps ofobtaining a cryoprecipitate containing both fibronectin and Factor VIIIfrom blood plasma and dissolving the cryoprecipitate in an aqueoussolution containing heparin to provide a cryoprecipitate/heparinsolution which contains from about 30 to about 150 units of heparin permilliliter of solution. The fibronectin and Factor VIII concentrates arethen recovered from the cryoprecipitate/heparin solution.

In one embodiment of practice of the invention, PEG is added to thecryoprecipitate/heparin solution to provide a PEG concentration of fromabout 1% to about 5% to precipitate fibronectin, while leaving FactorVIII remaining in the solution. The fibronectin precipitate is recoveredand further processed to provide the fibronectin concentrate, and thesupernatant is processed to produce the Factor VIII concentrate.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features and advantages of the present invention willbecome better understood with reference to the following description,appended claims, and accompanying drawings, wherein:

FIG. 1 is a flow chart illustrating an exemplary embodiment of a processprovided in accordance with practice of the present invention forpreparing a fibronectin concentrate from blood plasma.

FIG. 2 is a flow chart illustrating an exemplary embodiment of a processprovided in accordance with practice of the present invention forpreparing a Factor VIII concentrate from blood plasma.

DETAILED DESCRIPTION OF THE INVENTION

Preferred embodiments of the process provided in accordance withpractice of principles of this invention for producing fibronectin andFactor VIII concentrates can be understood by referring to FIGS. 1 and2, respectively, which are flow diagrams illustrating the process steps.As is described below in greater detail, both the fibronectin and FactorVIII are recovered from the same starting material, i.e., fromcryoprecipitate. The process for the recovery of both begins with thesteps of their separation from a cryoprecipitate solution.

Separation of the Fibronectin and Factor VIII Components ofCryoprecipitate

Turning first to FIG. 1, the process for preparing fibronectin isoutlined, including the initial steps of separating the fibronectin fromFactor VIII. Fibronectin and Factor VIII are initially separated fromhuman blood plasma that has been collected and tested according toprocedures approved by the U.S. Food and Drug Administration. The plasmais frozen at a temperature of about -20° C. and is subsequently thawedat 0° C. to 5° C. and pooled. During the thawing process, a precipitateforms (hereinafter called the "cryoprecipitate") which is removed bycentrifugation and recovered for further purification and concentration.

The cryoprecipitate (which contains both the fibronectin and Factor VIIIfractions of plasma) is dissolved in distilled water which contains fromabout 30 to about 150 units of heparin per milliliter (ml) of water.(The heparin-containing distilled water solution is hereinafter calledthe "heparin solution"). The heparin solution, which has been adjustedto a pH of from about 6.5 to about 7.5 with dilute hydrochloric acid(HCL) (or other appropriate acid such as acetic acid), is then mixed ata temperature of from about 20° C. to about 30° C. until thecryoprecipitate is completely dissolved to provide acryoprecipitate/heparin solution. Preferably, the temperature duringmixing is maintained at about 30° C., and the volume of heparin solutionused is from about 2 to about 10 liters per kilogram of cryoprecipitate.

As used herein, one unit of heparin is defined to mean one U.S.P.(United States Pharmacopoeia) unit. The U.S.P. unit of heparin is thatquantity which will prevent 1.0 ml of citrated sheep plasma fromclotting for one hour after the addition of 0.2 ml of a 1:100 CaCl₂solution. As used herein, the term "heparin" is meant to include heparinitself and the pharmaceutically-acceptable water soluble salts ofheparin, e.g., the sodium salts. A suitable example of acommercially-available heparin sodium product is U.S.P. heparin fromLyphomed Company, of Melrose Park, Ill.

PEG powder, preferably having a molecular weight in the range of fromabout 2000 to about 6000 (more preferably, from about 3000 to 4000), isthen added to the cryoprecipitate/heparin solution to provide a firstPEG solution having a final PEG concentration of from about 1% to about5% (wt/vol). The term (wt/vol) means the weight of material added per100 ml of solution prior to the addition. The percentages used hereinare all weight per volume unless otherwise indicated. Preferably, thePEG is added in the form of a solution prepared by dissolving the PEG indistilled water that contains a citrate (such as sodium citrate). In oneexemplary embodiment, the aqueous PEG solution added to thecryoprecipitate/heparin solution contains about 31.5% PEG, 0.22% sodiumcitrate dihydrate and 0.08% citric acid monohydrate at a pH of 6.2.

The pH of the first PEG solution is adjusted to from about 5.5 to 7.1with an acid such as dilute acetic acid. As is described later indetail, the yield and specific activity of Factor VIII is enhanced whenthe pH is from about 5.9 to 6.2 and, thus, it is preferred that the pHis adjusted to within this range and, most preferably, to about 6.1. ThepH-adjusted first PEG solution is mixed for approximately 15 minutes,while being maintained at a temperature of from about 15° C. to about35° C.

The addition of PEG (1%-5%) to form the first PEG solution results inprecipitation of fibronectin, along with fibrinogen, leaving Factor VIIIin the solution. The fibronectin and other precipitated proteins, i.e.,the first PEG precipitate, are separated from the Factor VIII-containingsolution by centrifugation. The first PEG precipitate is recovered andprocessed further to recover fibronectin, while the supernatant (thefirst PEG supernatant) is recovered and processed further to recoverFactor VIII.

It has been found that the preferred mixing temperature of the first PEGsolution is 20° C. to 30° C., with the temperature most preferred at 25°C. It was discovered that at temperatures less than about 25° C., andmore particularly at temperatures less than about 20° C., Factor VIIIprecipitates with the fibronectin proteins to a greater degree thandesired, thus reducing the yield of Factor VIII and reducing thespecific activity of the fibronectin produced. At temperatures aboveabout 25° C., and more particularly above about 30° C., increasedbacterial growth and denaturation of protein occur.

While the range of the concentration of heparin in thecryoprecipitate/heparin solution can be from about 30 to about 150 unitsper ml, the preferred range is from about 40 to 100 units per ml and,most preferably, is about 80. At less than about 30 units per ml, anundesirably low amount of fibronectin is precipitated, while adding morethan 150 units per ml does not sufficiently increase the amount offibronectin precipitated to be economical. As is shown below in Example4, about 80 units per ml of heparin is considered optimum.

While the range of from about 1% to about 5% PEG can be used in thefirst PEG solution, it is preferred that the solution have a PEGconcentration of from about 3% to about 4% to maximize the precipitationof fibronectin. When the concentration of PEG in the solution is greaterthan about 5%, an undesirably large amount of Factor VIII isprecipitated with the fibronectin proteins, and, thus, the specificactivity of the final fibronectin product is reduced, as well as theyield of Factor VIII. If less than about 1% PEG is used, the amount offibronectin proteins precipitated is less than desired. Thus, by addingPEG to a concentration of from about 1% to about 5%, the PEGconcentration is high enough to precipitate fibronectin, but is belowthe concentration at which Factor VIII will precipitate.

Production of Fibronectin from First PEG Precipitate

The first PEG precipitate is dissolved in distilled water in an amountcomprising from about 6 to about 20 times the total weight of theprecipitate, to thereby form the first PEG precipitate solution. Glycineis added to the solution to a final concentration of from about 1 toabout 1.8 moles per liter, i.e., from about 1 to about 1.8 molar (M)glycine. The pH of the solution is adjusted to between about 6.4 andabout 7.4. In an exemplary embodiment, the pH is adjusted to 6.7. Thetemperature of the first PEG precipitate solution is maintained,preferably, between about 20° C. to about 30° C. In an exemplaryembodiment, the temperature is maintained at 25° C. Various proteins,such as fibrinogen are precipitated from the first PEG precipitatesolution by the addition of glycine. The solution is then centrifuged,the precipitate is discarded, and the supernatant (the first PEGsupernatant), which contains fibronectin, is recovered.

PEG is added to the first PEG supernatant to a final PEG concentrationof from about 4% to about 12% to provide a second PEG solution. As wasthe case for the PEG added to form the first PEG solution, when solidPEG is used, it is preferred that the PEG is added in the form of asolution containing citrate, e.g., sodium citrate, at a concentration offrom about 0.01M to about 0.1M. Preferably, the concentration is about0.05M sodium citrate. When PEG is added in solid form, the water volumeused in dissolving the first PEG precipitate is preferably from about 10to about 20 times the weight of the precipitate. The addition of PEG tothe 4-12% concentration results in fibronectin precipitating from thesolution during mixing. Such mixing is done for not less than about 30minutes, at a temperature of from about 20° C. to about 30° C. Thefibronectin precipitate is recovered from the second PEG solution bycentrifugation.

The PEG added to form the second PEG solution preferably has a molecularweight in the range of from about 2000 to about 6000, more preferably inthe range of from about 3000 to about 4000. Additionally, during thesecond PEG precipitation step, it is preferred that the mixingtemperature be from about 20° C. to about 30° C., with the temperaturemost preferred at about 25° C.

Although the range of from about 4% to about 12% PEG in the second PEGsolution is satisfactory, the range of about 8% to about 10% ispreferred, with about 9% being most preferred. At less than about 8%,the amount of fibronectin precipitated is less than desired; at greaterthan about 12%, other proteins will precipitate, decreasing the specificactivity of fibronectin.

The PEG-precipitated fibronectin, i.e., the second PEG precipitate,which is recovered by centrifugation, is preferably subjected to twoidentical citrate/glycine washes in succession. In an exemplaryembodiment, the washes are accomplished by suspending the second PEGprecipitate in a solution having a glycine concentration of from about1M to about 1.8M and containing sodium citrate at a concentration offrom about 0.1M to about 0.5M. The pH of the solution is preferablyadjusted to 6.2 to 7.2 and is maintained at a temperature in the rangeof 5° C. to 25° C. After the suspension has been mixed thoroughly, e.g.,by mixing for 30 minutes to 1 hour, it is centrifuged to recover thefirst glycine-washed fibronectin precipitate. The volume of the glycinesolution is preferably from about 10 to about 30 liters per kilogram ofthe second PEG precipitate. The wash procedure is repeated to provide asecond citrate/glycine wash and the second, or final, glycine-washedfibronectin precipitate is recovered, e.g., by centrifugation.

The citrate/glycine washes remove immunoglobulins, Beta globulins, andimpurities from the fibronectin concentrate, as well as residual PEG.The citrate concentration preferred is from about 0.1M to 0.5M, with themost preferred range being from about 0.2M to 0.25M. It was found thatif the citrate concentration used was less than about 0.1M, theimmunoglobulins and Beta globulins were not removed as desired. If thecitrate concentration is greater than about 0.5M, no further removal ofimpurities occurs. The pH of about 6.7 is optimum, with a range of 6.2to 7.2 being satisfactory. If the temperature is greater than about 25°C., an undesirable amount of fibronectin will solubilize, therebyreducing the fibronectin yield.

After recovery, the glycine-washed fibronectin precipitate isreconstituted in an aqueous solution which, in one exemplary embodiment,includes 0.005M sodium citrate and 1% glycine. The reconstitutedfibronectin solution is then filtered, placed into final containers, andlyophilized.

In preferred embodiments of practice of principles of this invention,the fibronectin production process includes steps for inactivatingviruses that may be present in such blood products, e.g., hepatitis Bvirus, hepatitis non-A/non-B virus, HTLV III (AIDS virus),Cytomegalovirus, Epstein-Barr virus, and the like.

In one preferred embodiment of practice of the process of thisinvention, a solution comprising both an organic solvent and a detergentis added to the first PEG supernatant to inactivate virus that may bepresent. The amount of organic solvent and detergent added preferablyresults in a solution containing about 0.3% organic solvent and about 1%detergent. Detergents useful in practice of principles of the inventionare a detergent sold under the trademark "TWEEN-80" by Fisher Scientificof Springfield, N.J., or a detergent sold under the trademark "TRITONX-100" by Aldrich Company of Milwaukee, Wis. Useful organic solvents aretri(n-butyl)phosphate (TNBP) and ethyl ether, and the like. The solutionis incubated for about 6 hours, at a temperature of from about 24° C. toabout 30° C.

After the organic solvent/detergent solution inactivates virus presentin the protein being sterilized, the process is continued by adding PEGto the solution to thereby form the second PEG solution as describedabove, which results in precipitation of fibronectin. In other preferredembodiments, the organic solvent/detergent solution (the anti-viralsolution) can be added at another stage of the process instead of to thefirst PEG supernatant. For example, the anti-viral solution can be addedduring either of the citrate glycine wash steps, or it can be addedafter the fibronectin is dissolved in the 0.005M citrate/1% glycinesolution just prior to its being filled into the final containers.Inactivation of virus using organic solvent/detergent mixtures isdisclosed in U.S. Pat. No. 4,540,573 which issued on Sep. 10, 1985 toNeurath et al. U.S. Pat. No. 4,540,573 is incorporated herein by thisreference.

In a second preferred embodiment of practice of process of thisinvention, the steps applied to inactivate viruses can include heating,either alone or in combination with organic solvent/detergentinactivation. Such heat inactivation can, for example, include heatingthe fibronectin, after it is lyophilized, to at least about 60° C. for aperiod of time sufficient to inactivate the virus. The heating time canrange from 10 hours to 100 hours, as appropriate, and the heating can bedone with lyophilized fibronectin in bulk or in the final container.

One method that can be used in accordance with practice of the processof this invention for heating lyophilized fibronectin in bulk is todisperse (suspend) the lyophilized fibronectin in an organic liquid suchas heptane. The fibronectin suspension is then heated at not less thanabout 60° C. for not less than about 10 hours, preferably not less thanabout 24 hours. After the heating step is concluded, the suspension isfiltered, and the virus-inactivated fibronectin concentrate powder isrecovered and air-dried to remove residual organic liquid. Theair-dried, heat-treated fibronectin powder is then dissolved in adextrose solution and filtered through previously-sterilized,bacteria-retentive membrane or cartridge filters, filled into the finalcontainer (a clean, sterilized vial), and lyophilized.

Various methods for heat treating lyophilized proteins to inactivatevirus by suspending the proteins in organic liquids are disclosed inU.S. Pat. No. 4,490,361, which issued to C. Heldebrant on Dec. 25, 1984.U.S. Pat. No. 4,490,361 is incorporated herein by this reference.

The lyophilized fibronectin product produced in accordance with practiceof this invention can be used for intravenous injection for treatingtrauma, burn injury, sepsis, or malnutrition after reconstitution in anappropriate amount of sterile water. In one example for treating severemalnutrition, the reconstituted fibronectin is administered to patientsfor 4 days at doses of from about 7 to about 21 mg of fibronectin per kgof body weight.

Various features and advantages of the process for producing fibronectinin accordance with practice of this invention are illustrated in thefollowing examples. It is to be understood, however, that the examplesmerely illustrate the invention and are not intended to limit the scopeof the invention, which is defined by the claims.

EXAMPLE 1 Preparation of Fibronectin Concentrate

410 grams of cryoprecipitate were dissolved in distilled water whichcontained about 80 units of heparin per milliliter at a pH of 7. Thesolution was mixed at a temperature of about 30° C. for about 30 minutesto provide a cryoprecipitate/heparin solution.

A PEG solution having a PEG concentration of 31.5%, 0.22% sodium citratedihydrate, and 0.08% citric acid monohydrate at a pH of 6.2 was added tothe cryoprecipitate/heparin solution to provide a PEG concentration of3.5%. The resulting solution was mixed for 30 minutes at a temperatureof 25° C. A precipitate was formed (the first PEG precipitate) which wasseparated from the solution by centrifugation. The first PEG precipitatewas found to contain fibronectin, fibrinogen and immunoglobulins. Thesupernatant which contained Factor VIII was set aside.

155 grams of the first PEG precipitate were dissolved in distilled waterin an amount which comprised about 8 times the total weight of theprecipitate to form the first PEG precipitate solution. Glycine wasadded to the solution to a final concentration of 1.7M, and the pH ofthe solution was adjusted to about 6.6. The temperature of the solutionwas maintained at about 25° C., and the solution was centrifuged, andthe supernatant, which contained fibronectin, was recovered.

The PEG solution was added to 1260 ml of the supernatant to provide asolution with a PEG concentration of about 9% (the second PEG solution).The second PEG solution was mixed for approximately 30 minutes at atemperature of about 25° C., during which time a precipitate formed (thesecond PEG precipitate). The solution was centrifuged, and theprecipitate was recovered.

The second PEG precipitate was then subjected to two identicalcitrate/glycine washes in succession. 48 grams of the second PEGprecipitate were suspended in a solution having a glycine concentrationof about 1.8M and containing sodium citrate at a concentration of about0.25M. The pH of the solution was adjusted to 6.7 and maintained at atemperature of 20° C. After the suspension was mixed thoroughly bymixing for 30 minutes, it was centrifuged to recover the firstglycine-washed fibronectin precipitate. The citrate wash procedure wasthen repeated.

The glycine-washed fibronectin precipitate was reconstituted in anaqueous solution of 0.005M sodium citrate and 1% glycine to provide afinal bulk solution. The solution was then filtered, placed into finalcontainers, and lyophilized.

It was found that the final fibronectin product was 100% pure by acellulose acetate electrophoresis process, i.e., it was devoid offibrinogen and immunoglobulin impurities.

EXAMPLE 2 Monkey Kidney Cell Adhesion to Fibronectin

A portion of a fibronectin sample prepared according to the process ofExample 1 was tested for biological functional cell attachment assay.The fibronectin sample supported the MK2 (Rhesus monkey kidney) cellattachment on microtiter wells at very low concentrations (0.3micrograms fibronectin/ml).

EXAMPLE 3 Characterization of Fibronectin by the Process of ThisInvention

A portion of a fibronectin sample prepared according to the process ofExample 1 was analyzed for molecular weight protein patterns underreduced and non-reduced conditions using 5% sodium dodecyl sulfate (SDS)electrophoresis in conjunction with immunoblotting analysis (Westernblotting). (The procedures for the SDS and Western blotting analysis areoutlined in Blumenstock, F. et al, "Progressive Loss of Fibronectin -Mediated Opsonic Activity in Plasma Cryoprecipitate with Storage," VoxSang, 54, pp. 129-137 (1988).)

The fibronectin sample shows greater than 90% fibronectin dimer(supported by protein staining and immunoblotting analysis) and isdevoid of low molecular weight protein impurities. A complete conversionof fibronectin dimer into fibronectin monomer subunits occurs uponreduction by 10 mM beta-mercaptoethanol.

Having a fibronectin product of high purity which is non-degraded andnon-fragmented, and thus which has a relatively high dimer content,i.e., greater than 90% dimer, may provide enhanced in vivo activity andresponse.

EXAMPLE 4 Effect of Varying Concentrations of Heparin on the Separationof Fibronectin from Cryoprecipitate

250 grams of cryoprecipitate were divided into five 50-gram portions.Each 50-gram portion was dissolved at 30° C. in 150 grams of watercontaining various amounts of heparin. The concentration of heparin inthe five solutions was 0, 13, 53, 80 and 133 units of heparin permilliliter of solution (u/ml).

The pH of each of the five solutions was adjusted to approximately 7,using 0.1 normal (N) hydrochloric acid (HCl). A PEG solution containing0.01M sodium citrate and 0.004M citric acid was added to each solutionat 25° C. to provide a final PEG concentration of approximately 3.5%.The pH of each solution was adjusted to about 6.1, using 1N acetic acid.

The suspensions were centrifuged at 25° C., and the precipitates werecollected and each was dissolved in distilled water using a ratio of 1gram of precipitate to 6 grams of distilled water. The pH of eachsolution was adjusted to 6.8 by the addition of 1N acetic acid, andglycine was added to each solution at 20° C. to provide solutions havinga 1.8M glycine concentration. The suspensions were then centrifuged, andthe supernatants collected. The supernatants were assayed forfibronectin content by cellulose acetate electrophoresis and RID (radialimmuno diffusion) methods.

Table 1 below sets forth the results of the fibronectin assays of eachof the five solutions, in units of milligrams of fibronectin per literof plasma (mg/L).

                  TABLE 1                                                         ______________________________________                                                           Fibronectin    Fibronectin                                 Soln.                                                                              Heparin       mg/L           mg/L                                        No.  (in cryo solution)                                                                          (by electrophoresis)                                                                         (by RID)                                    ______________________________________                                        1    0        u/ml     37           55                                        2    13                50           62                                        3    53                75           69                                        4    80                81           82                                        5    133               86           89                                        ______________________________________                                    

As can be seen by the results of the experiments of Example 4, theamount of fibronectin precipitated from solution increased as the amountof heparin in the solution increased. For example, the amount offibronectin precipitated when 80 u/ml of heparin are used, based on theRID analysis, is 132% of the amount of fibronectin precipitated whenonly 13 u/ml heparin are used. The difference is even greater when thedata from the electrophoresis method is correlated.

Production of Factor VIII from the First PEG Supernatant

The recovery of Factor VIII from the first PEG supernatant provided inaccordance with the process described above is accomplished by firstadding PEG to the first PEG supernatant to provide a second PEG solutionwith a PEG concentration of from about 8% to about 16%. As was the casefor the PEG added to form the first PEG solution, it is preferred thatthe PEG is added in the form of a solution containing citrate, e.g.,sodium citrate, at a concentration of from about 0.01M to about 0.1M.The addition of PEG to the 8-16% concentration results in Factor VIIIprecipitating from the second PEG solution during mixing, which iscontinued for not less than about 15 minutes at a temperature of from20° C. to 30° C. In an exemplary embodiment, the Factor VIII precipitateis recovered from the second PEG solution by centrifugation.

The PEG added to form the second PEG solution preferably has a molecularweight in the range of from about 2000 to about 6000, more preferably inthe range of from about 3000 to about 4000. Additionally, duringprecipitation of Factor VIII from the second PEG solution, it ispreferred that the mixing temperature be from about 15° C. to about 30°C., with the temperature most preferred at about 25° C. It wasdiscovered that at temperatures less than about 15° C., and moreparticularly at temperatures less than about 10° C., protein impuritieswill precipitate along with Factor VIII, thereby reducing the specificactivity of the Factor VIII in the final product.

Although the range of from about 8% to about 16% in the second PEGsolution is satisfactory, the range of about 9% to about 15% ispreferred, with about 12% being most preferred. At less than about 8%,the precipitated Factor VIII yield is less than desired; at greater thanabout 16%, unwanted proteins will precipitate with Factor VIII, therebyreducing the specific activity of Factor VIII in the final product.

The Factor VIII precipitated from the second PEG solution is preferablysubjected to two identical citrate/glycine washes in succession. In anexemplary embodiment, the washes are accomplished by suspending thePEG-precipitated Factor VIII in a 1M to 2M glycine solution, whichincludes from about 10 to about 20 units of heparin per ml of solution,about 0.25M citrate, has a pH of about 6.7, and is maintained at atemperature in the range of 0° C. to 15° C. If desired, heparin can beomitted. It is preferred, however, that heparin is used to provideenhanced stability to the Factor VIII if a viral inactivation stepcomprising heat is performed subsequent to the wash steps. After thesuspension has been mixed thoroughly, e.g., by mixing for about 30 toabout 60 minutes, it is centrifuged to recover the glycine-washed FactorVIII precipitate. The volume of the glycine solution is preferably fromabout 5 to about 35 liters per kilogram of PEG-precipitated Factor VIII.The wash procedure is repeated to provide a second citrate/glycine washand the final glycine-washed Factor VIII precipitate is recovered, e.g.,by centrifugation.

The citrate/glycine washes remove immunoglobulins and Beta globulinsfrom the Factor VIII concentrate, as well as residual PEG. The citrateconcentration preferred is from about 0.1M to 0.5M, with the mostpreferred range being from about 0.2M to 0.25M. It was found that if thecitrate concentration used was less than about 0.1M, the immunoglobulinsand Beta globulins were not removed as desired. If the citrateconcentration is greater than about 0.5M, no further benefit isobserved. The pH of 6.7 is optimum, with a range of 6.2 to 7.2 beingsatisfactory. Preferably, the washes are done at less than 15° C. tominimize the amount of Factor VIII dissolved in the wash solution.

After recovery, the glycine-washed Factor VIII precipitate isreconstituted in an aqueous solution which, in one exemplary embodiment,includes 0.005M citrate and 1% glycine. The solution is then filtered,placed into final containers, and lyophilized.

In preferred embodiments of practice of principles of this invention,the process includes steps for inactivating viruses that may be presentin the blood products, as was described above for the fibronectinprocess. In one preferred embodiment of practice of the process of thisinvention, a solution comprising an organic solvent and a detergent isadded to the first PEG supernatant to inactivate virus that may bepresent. The amounts and types of organic solvents and detergents arethe same as those described above for viral inactivation in thefibronectin process.

After the organic solvent/detergent solution inactivates virus presentin the protein being sterilized, the process is continued as set forthabove by adding PEG to thereby form the second PEG solution toprecipitate Factor VIII. In other preferred embodiments, the organicsolvent/detergent solution (the anti-viral solution) can be added atanother stage of the process instead of to the first PEG supernatant.For example, the anti-viral solution can be added during the citrateglycine wash, or it can be added after the Factor VIII is dissolved inthe 0.005M citrate/1% glycine solution just prior to its being filledinto the final containers.

As was described above with regard to fibronectin, in a second preferredembodiment of practice of process of this invention to produce FactorVIII, the steps applied to inactivate viruses can include heating,either alone or in combination with organic solvent/detergentinactivation. The procedures used for Factor VIII are the same as thosedescribed above for fibronectin. Thus, one method that can be used forheating lyophilized Factor VIII in bulk, is to disperse (suspend) thelyophilized Factor VIII in an organic liquid such as heptane. The FactorVIII suspension is then heated at not less than about 60° C. for notless than about 10 hours, preferably not less than about 24 hours. Afterthe heating step is concluded, the suspension is filtered, and thevirus-inactivated Factor VIII concentrate powder is recovered andair-dried to remove residual organic liquid. The air-dried, heat-treatedFactor VIII powder is then dissolved in a dextrose solution and filteredthrough previously-sterilized, bacteria-retentive membrane or cartridgefilters, filled into the final container (a clean, sterilized vial), andlyophilized.

The lyophilized Factor VIII product produced in accordance with practiceof this invention can be used for intravenous injection to treathemophilia after reconstitution in an appropriate amount of sterilewater.

Examples of processes provided in accordance with this invention forproduction of Factor VIII are set forth below.

EXAMPLE 5

Human plasma (Type O) was initially frozen at a temperature of about-20° C. The frozen plasma was then thawed at from 0° C. to 5° C. andpooled. The cryoprecipitate which formed during the thawing process wasthen removed by centrifugation and recovered.

100 grams of cryoprecipitate was dissolved in 300 ml of distilled water,and liquid U.S.P. heparin from Lyphomed Company was added to provide acryoprecipitate/heparin solution containing 2.7 units of heparin per mlof solution. The solution was adjusted to a pH of 7 with 0.1N HCl andwas then mixed at 30° C. for 30 minutes.

Solid PEG powder, having a molecular weight of 3500 was provided in asolution containing about 0.05M sodium citrate. A portion of the PEGsolution was added to the cryoprecipitate/heparin solution to provide afirst PEG solution with a final PEG concentration of 3.5%. The first PEGsolution was thoroughly mixed at 25° C. for 15 minutes. The pH of thesolution was adjusted to 6.3, with dilute acetic acid, and the solutionwas then mixed at about 25° C. for about 30 minutes to precipitateunwanted proteins from the Factor VIII. The precipitate was removed bycentrifugation and saved for further processing into a fibronectinconcentrate. The supernatant (the first PEG supernatant), whichcontained the Factor VIII, was retained for further processing into asecond portion of the Factor VIII concentrate.

The PEG solution was added to the first PEG supernatant to bring theconcentration of PEG to 12%. The increased concentration of PEG resultedin precipitation of Factor VIII, and the suspension containing theFactor VIII precipitate was mixed for 15 minutes at 25° C. The FactorVIII precipitate was recovered by centrifugation and subjected to twoidentical glycine/citrate washes in succession. The supernatant wasdiscarded. The glycine/citrate washes included suspending the FactorVIII precipitate in a 1.8M glycine solution containing 14 units ofheparin per ml of solution, 0.25M sodium citrate, and a pH of about 6.7.The temperature of the suspension was maintained at 5° C. during mixing,which took approximately 30 minutes. The suspension was then centrifugedand the Factor VIII precipitate recovered. The glycine/citrate washprocedure was repeated.

The glycine-washed Factor VIII precipitate was recovered bycentrifugation and was reconstituted in an aqueous solution of 0.005Mcitrate and 1% glycine at a pH of 7.25 to provide the final bulksolution.

The yield of Factor VIII activity in the final bulk was measured by APTTone-stage assay and found to be 170 units (Factor VIII activity) perkilogram of plasma, with a final bulk yield of 54%. (The procedure forthe APTT one-stage assay is outlined in Langdell, R. et al, Journal ofLab. Clin. Med., Vol. 41, pp. 637-647 (1953).) The final bulk yield isdefined as the total Factor VIII activity in the final bulk divided bythe total Factor VIII activity in the cryoprecipitate. The specificactivity of the Factor VIII product was found to be 5.6 units of FactorVIII activity per mg of protein recovered.

EXAMPLE 6

The procedure of Example 5 was repeated except that sufficient heparinwas added to the cryoprecipitate solution to provide acryoprecipitate/heparin solution which contained 67 units of heparin perml of solution.

The yield of Factor VIII activity in the final bulk was measured andfound to be 180 units per kilogram of plasma, with a final bulk yield of53%. The specific activity of the Factor VIII product was found to be6.4 units of Factor VIII activity per mg of protein recovered.

EXAMPLE 7

The procedure of Example 5 was repeated except that sufficient heparinwas added to the cryoprecipitate solution to provide acryoprecipitate/heparin solution which contained 73 units of heparin perml of solution.

The yield of Factor VIII activity in the final bulk was measured andfound to be 272 units per kilogram of plasma, with a final bulk yield of65%. The specific activity of the Factor VIII product was found to be8.9 units of Factor VIII activity per mg of protein recovered.

EXAMPLE 8

The procedure of Example 5 was repeated except that sufficient heparinwas added to the cryoprecipitate solution to provide acryoprecipitate/heparin solution which contained 80 units of heparin perml of solution.

The yield of Factor VIII activity in the final bulk was measured andfound to be 251 units per kilogram of plasma, with a final bulk yield of63%. The specific activity of the Factor VIII product was found to be12.7 units of Factor VIII activity per mg of protein recovered.

EXAMPLE 9

The procedure of Example 8 was repeated. The yield of Factor VIIIactivity in the final bulk was measured and found to be 217 units perkilogram of plasma, with a final bulk yield of 69%. The specificactivity of the Factor VIII product was found to be 13.9 units of FactorVIII activity per mg of protein recovered.

EXAMPLE 10

A control was run using the same procedure as Example 5 except that noheparin was added to the distilled water in which the cryoprecipitatewas dissolved.

The yield of Factor VIII activity in the final bulk was measured andfound to be 199 units per kilogram of plasma, with a final bulk yield of65%. The specific activity of the Factor VIII product was found to be5.6 units of Factor VIII activity per mg of protein recovered.

Table 2 lists the results of a Examples 5 through 10.

                  TABLE 2                                                         ______________________________________                                                        Final Bulk          Specific                                                  Yield (Units        Activity                                                  Factor VIII         (Units                                           Units/ml Activity/kg         Factor VIII                                      Heparin  Total      Factor VIII                                                                            Activity/mg                                      In       Recovered  Yield at Total                                     Example                                                                              Solution Precipitate)                                                                             Final Bulk*                                                                            Protein)                                  ______________________________________                                        5       2.7     170        54%      5.6                                       6      67.0     180        53%      6.4                                       7      73.0     272        65%      8.9                                       8      80.0     251        63%      12.7                                      9      80.0     217        69%      13.9                                      10     (0)      199        65%      5.6                                       ______________________________________                                         *Final Bulk Yield = Units of Factor VIII in final bulk per units of Facto     VIII in cryoprecipitate.                                                 

As is shown in Examples 5-10, the specific activity of the Factor VIIIconcentrate was unexpectedly significantly increased when greater thanabout 80 units of heparin was used per ml of the cryoprecipitatesolution. The specific activity was the same for the control (Example10) where no heparin was used and for Example 5 where 2.7 units ofheparin per ml were used.

Examples 11-13 were conducted using the same procedure as Example 5except that Type A plasma was used.

EXAMPLE 11

The procedure of Example 5 was repeated except that Type A plasma wasused instead of Type O, and sufficient heparin was added to thecryoprecipitate solution to provide a cryoprecipitate/heparin solutionwhich contained 1.7 units of heparin per ml of solution.

The yield of Factor VIII activity in the final bulk was measured andfound to be 186 units per kilogram of plasma, with a final bulk yield of34%. The specific activity of the Factor VIII product was found to be6.7 units of Factor VIII activity per mg of protein recovered.

EXAMPLE 12

The procedure of Example 11 was repeated except that thecryoprecipitate/heparin solution contained 80 units of heparin per ml ofsolution.

The yield of Factor VIII activity in the final bulk was measured andfound to be 306 units per kilogram of plasma, with a final bulk yield of66%. The specific activity of the Factor VIII product was found to be11.4 units of Factor VIII activity per mg of protein recovered.

EXAMPLE 13

The procedure of Example 12 was repeated. The yield of Factor VIIIactivity in the final bulk was measured and found to be 261 units perkilogram of plasma, with a final bulk yield of 47%. The specificactivity of the Factor VIII product was found to be 13.2 units of FactorVIII activity per mg of protein recovered.

EXAMPLE 14

The procedure of Example 11 was repeated except that thecryoprecipitate/heparin solution contained 273 units of heparin per mlof solution.

The yield of Factor VIII activity in the final bulk was measured andfound to be 156 units per kilogram of plasma, with a final bulk yield of38%. The specific activity of the Factor VIII product was found to be5.2 units of Factor VIII activity per mg of protein recovered.

Table 3 lists the results of Example 11-14.

                  TABLE 3                                                         ______________________________________                                                        Final Bulk          Specific                                                  Yield (Units        Activity                                                  Factor VIII         (Units                                           Units/ml Activity/kg         Factor VIII                                      Heparin  Total      Factor VIII                                                                            Activity/mg                                      In       Recovered  Yield at Total                                     Example                                                                              Solution Precipitate)                                                                             Final Bulk                                                                             Protein)                                  ______________________________________                                        11      1.7     186        34%      6.7                                       12     80.0     306        66%      11.4                                      13     80.0     261        47%      13.2                                      14     273.0    156        38%      5.2                                       ______________________________________                                    

Examples 15 and 16 were conducted using the same procedure as Example 5except that Type B plasma was used.

EXAMPLE 15

The procedure of Example 5 was repeated except that Type B plasma wassubstituted for Type O plasma, and the cryoprecipitate/heparin solutioncontained 1.7 units of heparin per ml of solution.

The yield of Factor VIII activity in the final bulk was measured andfound to be 312 units per kilogram of plasma, with a final bulk yield of54%. The specific activity of the Factor VIII product was found to be6.3 units of Factor VIII activity per mg of protein recovered.

EXAMPLE 16

The procedure of Example 15 was repeated except that the heparinsolution contained 80 units of heparin per ml.

The yield of Factor VIII activity in the final bulk was measured andfound to be 349 units per kilogram of plasma, with a final bulk yield of61%. The specific activity of the Factor VIII product was found to be12.7 units of Factor VIII activity per mg of protein recovered.

Table 4 lists the results of Examples 15 and 16.

                  TABLE 4                                                         ______________________________________                                                        Final Bulk          Specific                                                  Yield (Units        Activity                                                  Factor VIII         (Units                                           Units/ml Activity/kg         Factor VIII                                      Heparin  Total      Factor VIII                                                                            Activity/mg                                      In       Recovered  Yield at Total                                     Example                                                                              Solution Precipitate)                                                                             Final Bulk                                                                             Protein)                                  ______________________________________                                        15     1.7      312        54%      5.3                                       16     80.0     349        61%      12.7                                      ______________________________________                                    

Examples 17-22 were conducted to show the effect of pH on both the finalbulk yield and specific activity of the Factor VIII product.

EXAMPLE 17

The procedure of Example 5 was repeated except that the cryoprecipitatesolution contained 80 units of heparin per ml, and the pH of the firstPEG solution was adjusted to 5.5 with dilute 1M acetic acid.

The yield of Factor VIII activity in the final bulk was measured andfound to be approximately 30 units per kilogram of plasma, and thespecific activity of the Factor VIII product was found to be about 2units of Factor VIII activity per mg of protein recovered.

EXAMPLE 18

The procedure of Example 17 was repeated except that the pH of the firstPEG solution was adjusted to 5.75.

The yield of Factor VIII activity in the final bulk was measured andfound to be approximately 145 units per kilogram of plasma, and thespecific activity of the Factor VIII product was found to be about 13units of Factor VIII activity per mg of protein recovered.

EXAMPLE 19

The procedure of Example 17 was repeated except that the pH of the firstPEG solution was adjusted to 6.0.

The yield of Factor VIII activity in the final bulk was measured andfound to be approximately 200 units per kilogram of plasma, and thespecific activity of the Factor VIII product was found to be about 20units of Factor VIII activity per mg of protein recovered.

EXAMPLE 20

The procedure of Example 17 was repeated except that the pH of the firstPEG solution was adjusted to 6.1.

The yield of Factor VIII activity in the final bulk was measured andfound to be approximately 248 units per kilogram of plasma, and thespecific activity of the Factor VIII product was found to be about 27units of Factor VIII activity per mg of protein recovered.

EXAMPLE 21

The procedure of Example 17 was repeated except that the pH of the firstPEG solution was adjusted to 6.15.

The yield of Factor VIII activity in the final bulk was measured andfound to be approximately 220 units per kilogram of plasma, and thespecific activity of the Factor VIII product was found to be about 11units of Factor VIII activity per mg of protein recovered.

EXAMPLE 22

The procedure of Example 17 was repeated except that the pH of the firstPEG solution as adjusted to 6.35.

The yield of Factor VIII activity in the final bulk was measured andfound to be approximately 220 units per kilogram of plasma, and thespecific activity of the Factor VIII product was found to be about 7units of Factor VIII activity per mg of protein recovered.

Table 5 lists the results of Example 17-22.

                  TABLE 5                                                         ______________________________________                                                                          Specific                                                             Final Bulk                                                                             Activity                                                             Yield (Units                                                                           (Units                                             Units/ml          Factor VIII                                                                            Factor VIII                                        Heparin           Activity/kg                                                                            Activity/mg                                 Example                                                                              In Solution                                                                             pH      Plasma)  Total Protein)                              ______________________________________                                        17     80        5.50     30      2.0                                         18     80        5.75    145      13.0                                        19     80        6.00    200      20.0                                        20     80        6.10    248      27.0                                        21     80        6.15    220      11.0                                        22     80        6.35    220      7.0                                         ______________________________________                                    

It can be seen that the yield and specific activity of the final FactorVIII product is increased when the pH of the first PEG solution isbetween about 6.0 to about 6.2. Preferably, the pH is about 6.1.

EXAMPLE 23

The procedure of Example 5 was repeated except that the cryoprecipitatesolution contained 80 units of heparin per ml of solution, and theliquid used to provide both the 3.5% and 12% PEG solutions contained0.011M citrate.

The percent yield of Factor VIII in the final bulk was measured at 62%with a specific activity of 5 units per mg of protein.

EXAMPLE 24

The procedure of Example 23 was repeated except that the PEG solutionwas added without citrate.

The percent yield of Factor VIII in the final bulk was measured at 38%,and the specific activity was 3.8 units of Factor VIII activity per mgof protein.

As can be seen from Examples 23 and 24, having citrate present in theliquid PEG solution increases both the percent final bulk yield andspecific activity of Factor VIII in the final product.

The Examples 25 through 32 compare the yield of the final Factor VIIIproduct when using different pH and PEG concentrations in the first PEGsolutions but maintaining both the first and second PEG solutions at 25°C.

EXAMPLE 25

The procedure of Example 5 was repeated except that the PEGconcentration in the first PEG solution was 3.3%, and the pH of thesolution was adjusted to 6.5. The specific activity of the Factor VIIIproduced was 3.9, and the final bulk yield was 50%.

EXAMPLE 26

The procedure of Example 5 was repeated except that the PEGconcentration in the first PEG solution was 3.5%, and the pH of thesolution was adjusted to 6.5. The specific activity of the Factor VIIIproduced was 5, and the final bulk yield was 50%.

EXAMPLE 27

The procedure of Example 5 was repeated except that the PEGconcentration in the first PEG solution was 3.5%, and the pH of thesolution was adjusted to 6.35. The specific activity of the Factor VIIIproduced was 12, and the final bulk yield was 65%.

EXAMPLE 28

The procedure of Example 5 was repeated except that the PEGconcentration in the first PEG solution was 3.6%, and the pH of thesolution was adjusted to 6.5. The specific activity of the Factor VIIIproduced was 5.9, and the final bulk yield was 50%.

EXAMPLE 29

The procedure of Example 5 was repeated except that the PEGconcentration in the first PEG solution was 3.6%, and the pH of thesolution was adjusted to 6.6. The specific activity of the Factor VIIIproduced was 4.4, and the final bulk yield was 45%.

EXAMPLE 30

The procedure of Example 5 was repeated except that the PEGconcentration in the first PEG solution was 3.8%, and the pH of thesolution was adjusted to 6.35. The specific activity of the Factor VIIIproduced was 11.5, and the final bulk yield was 26%.

EXAMPLE 31

The procedure of Example 5 was repeated except that the PEGconcentration in the first PEG solution was 4%, and the pH of thesolution was adjusted to 6.8. The specific activity of the Factor VIIIproduced was 1.9, and the final bulk yield was 40%.

Table 6 lists the results of Example 25-31.

                  TABLE 6                                                         ______________________________________                                                                 Specific Activity                                                             (Units of Factor                                            % PEG In          VIII Activity                                               First PEG         Per Mg of  Bulk Yield                                Example                                                                              Solution  pH      Total Protein)                                                                           %                                         ______________________________________                                        25     3.3       6.5     3.9        50                                        26     3.5       6.5     5.0        50                                        27     3.5       6.35    12.0       65                                        28     3.6       6.5     5.9        50                                        29     3.6       6.6     4.4        45                                        30     3.8       6.35    11.5       26                                        31     4.0       6.8     1.9        40                                        ______________________________________                                    

It can be seen that the optimum conditions for recovery and specificactivity of Factor VIII from a PEG solution processed at 25° C. appearto be achieved when the PEG concentration is approximately 3.5%.

EXAMPLE 32 Comparison of the Amount of Factor VIII Precipitated fromCryoprecipitate When Using Heparin Alone Versus Using Heparin Plus PEGfor Such Precipitation

Three 100-gram samples of cryoprecipitate were obtained. In the processwhere heparin alone was used to precipitate proteins from the solution,one of the 100-gram samples of cryoprecipitate was dissolved in 20 mMtris (3:1) buffer solution, and 80 u/ml of heparin was added at 25° C.and at a pH of approximately 6.8. The resulting suspension wascentrifuged at 25° C., and the Factor VIII present in the supernatantwas concentrated into a precipitate by adding PEG to a finalconcentration of 12%. The Factor VIII precipitate was dissolved in a0.01M sodium citrate solution, using a ratio of 20 grams of water pergram of precipitate, and the solution was filtered. Assays wereconducted on the solution to determine the amount of fibrinogen, totalprotein, and the Factor VIII yield.

In the process where both heparin and PEG were used to precipitateproteins from the solution, the second 100-gram sample ofcryoprecipitate was dissolved in a heparin solution containing 80 unitsof heparin per ml of solution at a pH of approximately 7. PEG was addedto the solution to bring the PEG concentration of the solution to 3.5%.The resulting suspension was centrifuged at 25° C., and the Factor VIIIpresent in the supernatant was concentrated into a precipitate by addingPEG to a final concentration of 12%. The Factor VIII precipitate wasdissolved in a 0.01M sodium citrate solution, using a ratio of 20 gramsof water per gram of precipitate, and the solution was filtered. Assayswere conducted on the solution to determine the amount of fibrinogen,total protein, and Factor VIII yield.

The third 100-gram sample was dissolved in 20 mM tris buffer solutionand was assayed for fibrinogen, total protein and Factor VIII yield.

The assays were conducted using cellulose acetate electrophoresis, andtotal protein concentration was determined at 280 nanometers.

Table 7 shows the concentrations of fibrinogen in mg per ml of solution,total protein in mg per ml of solution, and Factor VIII yield inunits/kg of plasma.

                  TABLE 7                                                         ______________________________________                                                                     Units of                                                   Total    Total     Factor VIII                                                Fibrinogen                                                                             Protein   Remaining                                                  Remaining                                                                              Remaining In Solution                                                In Solution                                                                            In Solution                                                                             (Per kg of                                                 (mg/ml)  (mg/ml)   Plasma                                           ______________________________________                                        Reconstituted                                                                             14.6 mg/ml 24.0 mg/ml                                                                              372 u/kg                                     cryoprecipitate                                                               After heparin                                                                              8.1 mg/ml 13.7 mg/ml                                                                              196 u/kg                                     precipitation:                                                                After heparin plus                                                                         2.0 mg/ml  8.9 mg/ml                                                                              213 u/kg                                     PEG precipitation:                                                            ______________________________________                                    

It can be seen from the results shown in Table 7 that using PEG incombination with heparin is much more effective in precipitating thefibrinogen and other impurities from the Factor VIII than using heparinalone for the separation.

The above descriptions of exemplary embodiments of processes forproducing fibronectin and Factor VIII concentrates are for illustrativepurposes. Because of variations which will be apparent to those skilledin the art, the present invention is not intended to be limited to theparticular embodiments described above. The scope of the invention isdefined in the following claims.

What is claimed is:
 1. A process for producing a Factor VIII concentratefrom blood plasma, the process comprising the steps of:(a) obtaining acryoprecipitate containing Factor VIII from blood plasma; (b) dissolvingthe cryoprecipitate in an aqueous solution containing heparin in anamount sufficient to provide a cryoprecipitate/heparin solutioncontaining from about 30 to about 150 units of heparin per milliliter ofsolution: (c) adding a sufficient amount of a precipitant consistingessentially of PEG to the cryoprecipitate/heparin solution whilemaintaining the solution at a temperature of from 20° C. to 30° C. toprecipitate protein contaminants, leaving a PEG supernatant containingFactor VIII; (d) recovering the PEG supernatant; and (e) recoveringFactor VIII from the PEG supernatant.
 2. The process of claim 1 whereinthe cryoprecipitate/heparin solution contains from about 35 to about 100units of heparin per milliliter of solution.
 3. The process of claim 1wherein the pH of the solution of step (c) is adjusted to from about 5.9to about 6.2.
 4. The process of claim 1 additionally comprising thesteps of:(a) adding PEG to the PEG supernatant to provide a PEGconcentration of from about 8% to about 16% to precipitate Factor VIII;(b) recovering and then washing the Factor VIII precipitate in anaqueous solution having a glycine concentration of from about 0.1M toabout 2.6M, a citrate concentration of from about 0M to about 0.5M andcontaining from about 10 to about 15 units per milliliter of heparin;and (c) recovering the washed Factor VIII precipitate by centrifugation.5. A Factor VIII concentrate produced by the process of claim
 1. 6. Theprocess of claim 1, wherein the cryoprecipitate/heparin solutioncontains from at least about 80 to about 150 units of heparin permilliliter of solution and the pH of the solution is adjusted to about6.3.
 7. A process for producing a Factor VIII concentrate from bloodplasma, the process comprising the steps of:(a) obtaining acryoprecipitate containing Factor VIII from the blood plasma; (b)dissolving the cryoprecipitate in an aqueous solution containing heparinin an amount sufficient to provide a cryoprecipitate/heparin solutioncontaining from about 30 to about 150 units of heparin per milliliter ofsolution; (c) adding a precipitant consisting essentially of PEG to thecryoprecipitate/heparin solution, while maintaining the solution at atemperature of from 20° C. to 30° C., to provide a first solution havinga PEG concentration of from about 1% to about 5% and adjusting the pH ofthe solution to from about 5.9 to about 6.2 to thereby precipitateunwanted protein contaminants from the solution to provide a first PEGsupernatant containing Factor VIII; (d) recovering the first PEGsupernatant; (e) adding an additional precipitant consisting essentiallyof PEG to the first PEG supernatant to bring the PEG concentration tofrom about 8% to about 16%, while maintaining the solution at atemperature of from about 15° C. to about 30° C., to thereby precipitateFactor VIII from the supernatant; (f) recovering and then washing theFactor VIII precipitate in an aqueous solution containing (1) glycine ata concentration of from about 1.0M to about 2.6M, (2) citrate, and (3)heparin at a concentration of from about 10 to about 15 units heparinper milliliter of solution, wherein the wash is maintained at less thanabout 15° C.; and (g) recovering the washed Factor VIII precipitate. 8.The process of claim 7 wherein during step (c) the first PEG solution ismaintained at a temperature of about 25° C. during precipitation ofunwanted protein contaminated.
 9. The process of claim 7 wherein duringstep (c) the pH of the first PEG solution is adjusted to about 6.1. 10.A factor VIII concentrate produced by the process of claim
 7. 11. Theprocess of claim 7, wherein the cryoprecipitate/heparin solutioncontains from at least about 80 to about 150 units of heparin permilliliter of solution and the pH of the solution is adjusted to about6.3.
 12. A process for producing a Factor VIII concentrate from bloodplasma, the process comprising the steps of:(a) obtaining acryoprecipitate containing Factor VIII from blood plasma; (b) dissolvingthe cryoprecipitate in an aqueous solution containing heparin in anamount sufficient to provide a cryoprecipitate/heparin solutioncontaining from about 30 to about 150 units of heparin per milliliter ofsolution; (c) adding a precipitant consisting essentially of PEG to thecryoprecipitate/heparin solution to provide a first solution having aPEG concentration of from about 1% to about 5% and adjusting the pH ofthe solution to from about 5.9 to about 6.2 to thereby precipitateunwanted protein contaminants from the solution to provide a first PEGsupernatant containing Factor VIII; (d) recovering the first PEGsupernatant; (e) adding additional precipitant consisting essentially ofPEG to the first PEG supernatant to bring the PEG concentration to fromabout 8% to about 16% to thereby precipitate Factor VIII from thesupernatant; (f) recovering and then washing the Factor VIII precipitatein an aqueous solution containing (1) glycine at a concentration of fromabout 1 to about 2.6M, (2) citrate at a concentration of from about 0.1to about 0.5M, and (3) heparin at a concentration of from about 10 toabout 15 units of heparin per milliliter of solution, wherein the washis maintained at less than about 15° C.; and (g) recovering the washedFactor VIII precipitate.
 13. A process for producing a Factor VIIIconcentrate from blood plasma, the process comprising the steps of:(a)obtaining a cryoprecipitate containing Factor VIII from blood plasma;(b) dissolving the cryoprecipitate in an aqueous solution containingheparin in an amount sufficient to provide a cryoprecipitate/heparinsolution containing about 80 units of heparin per milliliter ofsolution; (c) adding a precipitant consisting essentially of PEG to thecryoprecipitate/heparin solution to provide a first solution having aPEG concentration of about 3% to 4% and adjusting the pH of the solutionto about 6.3 to thereby precipitate unwanted protein contaminants fromthe solution to provide a first PEG supernatant containing Factor VIII;(d) recovering the first PEG supernatant; (e) adding additionalprecipitant consisting essentially of PEG to the first PEG supernatantto bring the PEG concentration to about 12% to thereby precipitateFactor VIII from the supernatant; (f) recovering and then washing theFactor VIII precipitate in an aqueous solution containing (1) glycine ata concentration of about 1.6M, (2) citrate at a concentration of about0.2M, and (3) heparin at a concentration of from about 10 to about 15units of heparin per milliliter of solution, wherein the wash ismaintained at less than about 15° C.; and (g) recovering the washedFactor VIII precipitate.